Isotachophoresis detection

Determination of mandelic acid in urine has been recommended as a biomaiker of exposure to ethylbenzene. Several methods can be used to determine mandelic acid in urine samples. These include derivatization of the acid and GC analysis (detection limit, 1.0 mg/L) isotachophoresis (detection limit, 0.04 mmol/L) and high-performance liquid chromatography (detection limit, 0.01 mmol/L) (WHO, 1996a). 

Detection methods for low molecular weight compounds in zone electrophoresis are identical with those used in flat bed chromatography. In isotachophoresis detection is done by special techniques that have been referred to in the respective chapter. Therefore in the following sections attention will be paid to detection of biopolymers (mainly to techniques used in gel electromigration techniques). 

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

Later, an improved system for C02 photofixation was reported by the same authors.164 The new system consisted of 6.5 x 1(T5 M tris(2,2 -bipyridine)ruthenium(II), Ru(bpy)3, as the photosensitive electron donor, methyl viologen (MV2+, 20 mM) as the electron acceptor, and triethanolamine (TEOA, 0.6 M) as a sacrificial electron donor in a C02-saturated aqueous solution (Fig. 18). Under irradiation with a 300-W high-pressure Hg lamp with a CuS04 chemical filter (A > 320 nm), formic acid, which was detected by isotachophoresis, was produced in quantum yields of ca. 0.01%. Recently, however, Kase et al.165 have repeated this experiment using a 13C02 tracer and have claimed that the formic acid obtained was produced not by C02 reduction but rather by oxidative cleavage of TEOA. 

The hyphenation of CE and NMR combines a powerful separation technique with an information-rich detection method. Although compared with LC-NMR, CE-NMR is still in its infancy it has the potential to impact a variety of applications in pharmaceutical, food chemistry, forensics, environmental, and natural products analysis because of the high information content and low sample requirements of this method [82-84]. In addition to standard capillary electrophoresis separations, two CE variants have become increasingly important in CE-NMR, capillary electrochromatography and capillary isotachophoresis, both of which will be described later in this section. 

Since both the bubble cell and the Z-cell need high resolution in order to observe the sensitivity increase, test whether you can avoid the use by a clever injection procedure such as sample stacking or transient ITP (isotachophoresis) instead. Further information on detection approaches is provided in Chapters 3, 5, and 15. 

Fanali et al. have described a capillary isotachophoresis method for the determination of procaine in pharmaceuticals [ 150]. The drug was determined in a 6 pL sample of solution (Spofa product, obtained from Czechoslovakia, and diluted 180-fold) by cationic isotachophoresis in the single column mode. The system used a PTFE capillary column (20 cm x 0.3 mm) and a conductivity detector. The separation was carried out at room temperature, at 50 pA (but switched to 25 pA during detection). 

Finally, when RPC methods are used in preparative studies with peptides, the opportunity routinely exists for subsequent analysis of the recovered fractions by a variety of analytical methods including high-speed RP-HPLC, HP-IEX, HP-HILIC, or HP-IMAC, zonal or micellar electrokinetic high-performance capillary electrophoresis (HP-CZE and MECK-CZE), capillary electrochromatography (CEC), or capillary isotachophoresis. The combination of the RPC information, drawn from the In k versus i > plots, with the data derived from on-line spectroscopic detection thus readily provides a comprehensive opportunity to assess the purity of an isolated peptide, many of the physicochemical features of the interaction, as well as a means to optimize the resolution in the RPC separation. 

Other applications include the online coupling of capillary isotachophoresis and CZE for the quantitative determination of flavonoids in Hypericum perforatum (Guttiferae) leaves and flowers. This method involved the concentration and preseparation of the flavonoid fraction before introduction into the CZE capillary. The limit of detection for quercetin 3-0-glycosides was 100 ng/ml. ... 

Electrophoresis is one of many electromigrational separation techniques which include isotachophoresis, immunoelectrophoresis and isoelectric focussing that have been used to separate various species on the basis of their different mobility in an electric field. These techniques can be used not only to achieve separations but also it is possible to identify the ligand bound to the metal. This can be done by comparing the isoelectric points, immunological behaviours, extent of mobilities or step heights of the sample constituents with those of well-characterised standards. A difficulty, however, is in the determination of the metal constituent itself. Except in the case of radioisotopes, the activities of which can be easily measured, non-radioactive elements can be detected only after further separation steps. 

Triethanolamine can be determined in metalworking and cutting fluids by gas chromatography-mass selective detection of silylated derivatives, by isotachophoresis, by capillary zone electrophoresis with indirect ultraviolet detection, and by spectrophotometry (Kenyon et al, 1993 Fernando, 1995 Schubert et al, 1996 Sollenberg, 1997) and in cosmetics and pharmaceuticals by ion-exclusion chromatography and by reversed-phase high performance liquid chromatography (Fukui et al, 1992 Maurer etal, 1996). 

J.E. Prest, S.J. Baldock, P.R. Fielden and B.J.T. Brown, Determination of metal cations on miniaturised planar polymeric separation devices using isotachophoresis with integrated conductivity detection, Analyst, 126 (2001) 433-437. 

M. Galloway and S.A. Soper, Contact conductivity detection of polymerase chain reaction products analyzed by reverse-phase ion pair microcapillary electrochromatography, Electrophoresis, 23 (2002) 3760-3768. M. Masar, M. Dankova, E. Olvecka, A. Stachurova, D. Kaniansky and B. Stanislawski, Determination of free sulfite in wine by zone electrophoresis with isotachophoresis sample pretreatment on a column-coupling chip, J. Chromatogr. A, 1026 (2004) 31-39. 

Another CE separation method that has been adapted to on-line NMR detection for trace level separations is capillary isotachophoresis [22]. In this case, after the separation, the analyte bands are slowly moved through the capillary until they lie directly within the coil. Precise positioning of the analyte bands in the NMR detection coil can be difficult. A recent enhancement is the use of several NMR detection coils on a single separation capillary [23], In this way, the first coil acts as a scout coil and is optimized for sensitivity (not necessarily linewidth) to locate the analyte band as it moves through the coil. After an analyte band is detected, the flow is stopped after the appropriate time-interval so that the analyte bands are now located in the second coil, which is used to acquire high-resolution NMR spectra. 

Mohamadi et al. [96] reported online preconcentration of human serum albumin (HSA) and its immunocomplex with a monoclonal antibody on-chip coupled to isotachophoresis. The sample injection, preconcentration, and separation were carried out continuously and controlled by a sequential voltage switching program. Preconcentration was carried out with on-chip nondenaturing gel electrophoresis in methylcellulose solution. Furthermore, the authors applied this method for immunoassay of HSA. The separation of HSA and its immunocomplex was achieved in 25 seconds in 1 cm of the microchannel with induced fluorescence detection at 7.5 pM. 

Capillary electrophoresis (CE) coupled to MS has the advantage of high resolution and soft ionization for biomolecules, which may be used to differentiate post-translational modifications and variants of intact proteins and oligonucleotides. Different modes of CE (capillary zone electrophoresis, capillary isoelectric focusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as online preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) are used to compensate for the restricted detection sensitivity of the CE methodology [77, 78]. 

Bodor, R., Madajova, V., Kaniansky, D., Masar, M., Johnck, M., Stanislawski, B., Isotachophoresis and isotachophoresis-zone electrophoresis separations of inorganic anions present in water samples on a planar chip with column-coupling separation channels and conductivity detection. J. Chromatogr. A 2001, 916(1-2), 155-165. 

Prest, J.E., Baldock, S.J., Fielden, P.R., Goddard, N.J., Treves-Brown, B.J., Bidirectional isotachophoresis on a planar chip with integrated conductivity detection. 

R. D., Brown, B.J.T., Vaireanu, D.I., A miniaturized planar isotachophoresis separation device for transition metals with integrated conductivity detection. Micro Total Analysis Systems 98, Proceedings pTAS 98 Workshop, Banff, Canada,... 

The length of the analyte zone in cITP is a quantitative parameter and is related to the concentration of the analyte in that zone. The height of the step is a quantitative parameter that is characteristic of the analyte, and it is directly proportional to analyte mobility. Conductivity detection is the detection system generally used for isotachophoresis, although UV detection is employed occasionally in commercial instrumentation. Figure 5.19 shows a typical electropherogram obtained using cITP with conductivity detection. 

The separation unit of the capillary isotachophoresis instrument used is shown in Fig. 13.1. A 0.85mm id capillary tube made of fluorinated ethylene propylene copolymer was used in the pre-separation (first) stage and a capillary tube of 0.30mm id made of the same material served for the separation in the second stage. Both tubes were provided with conductivity detection cells [18] and an ac conductivity mode of detection [15] was used for making the separations visible. 

Radioisotope detection of P, 14C, and Tc was reported by Kaniansky et al. (7,8) for isotachophoresis. In their work, isotachophoretic separations were performed using fluorinated ethylene-propylene copolymer capillary tubing (300 pm internal diameter) and either a Geiger-Mueller tube or a plastic scintillator/photomultiplier tube combination to detect emitted fi particles. One of their reported detection schemes involved passing the radiolabeled sample components directly through a plastic scintillator. Detector efficiency for 14C-labeled molecules was reported to be 13-15%, and a minimum detection limit of 0.44 nCi was reported for a 212 nL cell volume. 

In recent years several sensitive and specific methods for the simultaneous determination of ephedrine alkaloids in plant material have been published. These include thin-layer chromatography (292,426), gas chromatography (251), straight-phase and reversed-phase high-performance liquid chromatography (253, 255, 302, 355, 427), isotachophoresis (303, 356), and 13C-NMR (304). Resolution of enantiomeric alkaloids by HPLC has been achieved on chiral stationary phases (417, 418) or after derivatization with a chiral agent on an achiral stationary phase (419). Chromatographic separation and analytical detection of... 

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